Cancer Stem Cells in Glioblastoma Multiforme

Abstract

Aim: To identify and characterize cancer stem cells (CSC) in glioblastoma multiforme (GBM).

Methods: Four-micrometer thick formalin-fixed paraffin-embedded GBM samples from six patients underwent 3,3-diaminobenzidine (DAB) and immunofluorescent (IF) immunohistochemical (IHC) staining for the embryonic stem cell (ESC) markers NANOG, OCT4, SALL4, SOX2, and pSTAT3. IF IHC staining was performed to demonstrate co-expression of these markers with GFAP. The protein expression and the transcriptional activities of the genes encoding NANOG, OCT4, SOX2, SALL4, and STAT3 were investigated using Western blotting (WB) and NanoString gene expression analysis, respectively.

Results: DAB and IF IHC staining demonstrated the presence of a CSC population expressing NANOG, OCT4, SOX2, SALL4, and pSTAT3 with the almost ubiquitous presence of SOX2 and a relatively low abundance of OCT4, within GBM. The expression of NANOG, SOX2 and, pSTAT3 but, not OCT and SALL4, was confirmed by WB. NanoString gene analysis demonstrated transcriptional activation of NANOG, OCT4, SALL4, STAT3, and SOX2 in GBM.

Conclusion: This study demonstrated a population of CSCs within GBM characterized by the expression of the CSC markers NANOG, SALL4, SOX2, pSTAT3 and OCT4 at the protein and mRNA levels. The almost ubiquitous presence of SOX2 and a relatively low abundance of OCT4 would support the putative existence of a stem cell hierarchy within GBM.

Keywords: cancer; embryonic; expression; glioblastoma multiforme; hierarchy; stem cells.

Figure 1

Representative image of H&E stained slides showing the presence of GBM (A) and DAB IHC-stained slides of GBM tissue demonstrating expression of CSC markers (B–F). pSTAT3 [(B), brown] was expressed in the nuclei of tumor and endothelium of the microvessels throughout the sample. SALL4 [(C), brown] was predominantly expressed on the nuclei of the tumor cells, and the cytoplasm of the endothelial cells lining the microvessels. SOX2 [(D), brown] displayed strong nuclear staining of the tumor cells, and moderate cytoplasmic staining of the endothelium of the microvessels. Nuclear expression of NANOG [(E), pink/red] was observed on the tumor cells, and to the lesser extent on the endothelium of the microvessels. Nuclear and cytoplasmic staining of OCT4 [(F), brown] was observed in few tumor cells. Cell nuclei were counterstained with hematoxylin [(A–F), blue]. Original magnification: 400X.

Figure 2

Representative images of IF IHC-stained sections of GBM tissue for ESC markers. SOX2 [(A), red, arrows], NANOG [(B), red, arrows], and pSTAT3 [(C), red, arrows] all showed nuclear expression on GFAP+ tumor cells [(A–C), green]. OCT4 [(D), red, arrows] staining was scarce and solely cytoplasmic in GFAP+ tumor cells. SALL4 [(E), green] and SOX2 [(E), red] were co-expressed (arrows) in the nuclei of some tumor cells, with SALL4 also staining SOX2–negative cells. Cell nuclei were counterstained with 4′, 6′-diamidino-2-phenylindole [(A–E), blue]. Scale bars: 20 μm.

Figure 3

Western blots of five GBM tissue samples. OCT4 was not detected (A), pSTAT3 at ~90 kDa was found in three out of five samples (B), NANOG was present in all five samples with multiple bands at approximately 40 and 31 kDa (C). SOX2 was detected in four out of five samples with bands at approximately 45 and 38 kDa (D).

Figure 4

Relative expression of CSC mRNA transcripts in six GBM samples showing the presence of all 5 markers at varying levels. NANOG, OCT4, and SALL4 showed relatively low mRNA expression, while STAT3 and SOX2 displayed high levels of mRNA expression. Expression is depicted relative to the housekeeper GUSB.